Some aspects of the biogenesis of tRNA and mRNA from primary gene transcripts to their final functional form in both E. coli and human KB cells will be investigated. Studies of tRNA function in E. coli will also be undertaken through an investigation of codon-anticodon recognition in vivo and the role of rare nucleotides in protein synthesis. An attempt will be made to answer the following questions: a) How do change in the nucleotide sequence of precursor tRNAs affect their interaction with RNase P? b) Is the pathway of tRNA biogenesis in human cells analagous to that found in E. coli? c) Are endoribonucleases important in post-transcriptional regulation of gene expression in E. coli and human cells? e) Does message context play a major role in affecting suppression of amber and ochre nonsence mutations in vivo? f) Can the absence of any particular nucleotide modification of tRNATyr drastically affect its ability to carry out protein synthesis in vitro? The systems my colleagues and I have developed to study the biosynthesis of tRNA and the nature of endoribonucleases in E. coli and human cells will be extensively utilized. Enzymatically and genetically altered tRNA precursors will be used as substrates to follow aspects of tRNA biosynthesis and function in vitro and to detect new endoribonucleolytic activities in sub-cellular fractions. Certain mRNAs of both pro-and eucaryotic origin will also be used in the search for endoribonucleases. In vivo suppression tests of allelic amber and ochre mutations in various genes will be used to compare the efficiency of ochre and amber suppressors derived from E. coli tRNATyr.